Rapid molecular methods for identification of pathogens and characterization of resistance mechanisms are essential to clinical infectious disease diagnostics. Our work aims to develop novel applications of mass spectrometry for these problems in the clinical microbiology laboratory. Our approach involves both the identification of new bacterial protein markers and the development of assays to detect these markers using LC-MS/MS and other mass spectrometry technologies. Methods are being pursued for (1) Culture-free identification of pathogenic bacteria in primary specimens, (2) Strain-level classification of bacterial pathogens, and (3) Rapid resistance protein identification. Previously published work involved identification of a peptide marker for tracking a carbapenemase-carrying resistance plasmid that could be detected by MALDI-TOF mass spectrometry (Lau et al, JCM, 2014), followed by a clinical validation study demonstrating the method's utility in identifying KPC carbapenemase proteins in clinical isolates from NIH Clinical Center patients (Youn et al, JCM, 2016). Work done during the 2016 and 2017 fiscal years in collaboration with Anthony Suffredini's group in CCMD, NIHCC included the development of a genoproteomics approach for identifying strain-specific peptide markers based on LC-MS/MS profiling of tryptic peptides, using clinical Acinetobacter baumannii isolates as a model (Wang et al, Clinical Chemistry, 2016). This approach is based on in silico computational analysis to guide selection of informative, genome-specific tryptic peptides from LC-MS/MS peptide profiles, followed by experimental confirmation. An extension of these genoproteomic methods to the design of assays for the culture-free identification of bacteria in primary specimens was developed and published in Wang et al, Clinical Chemistry, 2017. This proof-of-concept study demonstrated that rapid (90 minute) diagnostic identification of bacterial pathogens in primary specimens may be possible for a variety of specimen types without culture. These approaches were further refined and applied to validate an approach to the direct identification of the KPC carbapenemase in clinical isolates. This was done by choosing three tryptic peptides unique to the KPC protein based on bioinformatics analysis and optimal LC-MS/MS spectral characteristics. These peptides were validated in a set of clinical isolates and their performance characteristics were published in Wang et al, Scientific Reports, 2017. This method allows a rapid (90 minute) diagnostic mass spectrometry assay for detecting the KPC carbapenemase protein in clinical isolates. Work done in the 2018 fiscal year and currently underway in collaboration with the Suffredini lab aims to validateLC-MS/MS assays to detect the OXA-48 and NDM families of carbapenemases in clinical isolates. A separate project has focused on a rapid LC-MS/MS approach to detect the MCR-1 protein in clinical isolates. Additional work aims to develop rapid LC-MS/MS approaches to differentiate clinical Burkholderia cepacia complex isolates. Separate work done during the 2018 fiscal year in collaboration with Vincent Munster in the Laboratory of Virology, NIAID and with Dan Chertow in the NIH Clinical Center validated strategies for bacterial identification using MALDI-TOF MS compatible with BSL-4 level inactivation protocols. These approaches allow rapid MALDI-TOF MS-based bacterial identification in the diagnostic and clinical management of patients with high containment viral infection.